Recombinant cell clones having increased stability and methods of making and using the same

ABSTRACT

Disclosed are a stable recombinant cell clones which are stable in serum- and protein-free medium for at least 40 generations, a biomass obtained by multiplying the stable cell clone under serum- and protein-free culturing conditions, and a method of preparing recombinant proteins by means of the biomass. Furthermore, the invention relates to a method of recovering stable recombinant cell clones.

CROSS-REFERENCES TO RELATED APPLICATIONS

This patent application is a divisional of U.S. Ser. No. 11/123,362,filed May 6, 2005 which is a continuation of U.S. Ser. No. 09/324,612,filed Jun. 2, 1999, now U.S. Pat. No. 6,475,725 which is acontinuation-in-part application of U.S. Ser. No. 09/100,253, filed Jun.19, 1998, now U.S. Pat. No. 6,100,061, all of which are hereinincorporated by reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not applicable.

BACKGROUND OF THE INVENTION

The present invention relates to a stable recombinant cell clone that isstable for at least 40 generations in serum- and protein-free medium, abiomass obtained by multiplying the stable cell clone under serum- andprotein-free culturing conditions, and a method of preparing recombinantproteins by means of the biomass. Furthermore, the invention relates toa method of recovering stable recombinant cell clones. Furthermore, theinvention relates to the production of a recombinant protein in a serum-and protein-free synthetic minimum medium.

Another aspect of the invention is a serum- and protein-free medium forculturing cells expressing a recombinant protein.

The preparation of recombinant proteins, in particular of biomedicalproducts, such as blood factors, is gaining in importance. To allow foran optimum growth of recombinant cells, serum is added to the medium inmost instances. Because of the high costs of serum and for avoidingpossible contamination's in the culturing medium by viral or molecularpathogens from the serum, a number of serum-free media have beendeveloped which, in particular, should not contain any additives ofbovine or human origin. In addition to the low risk of contaminating theprepared products with viral and molecular pathogens, the use of suchmedia in the preparation process also allows for a simpler purificationof the expressed proteins.

In most instances, recombinant cells are first cultured inserum-containing medium up to a high cell density, e.g. for a workingcell bank, and subsequently they are re-adapted to serum-free mediumduring the production phase.

Miyaji et al., Cytotechnology, 3:133-140 (1990) selectedserum-independent cell clones in serum-free medium which containedinsulin and transferrin. However, the living cell number and theexpression rate proved to decrease continuously after 16 days. Byco-amplification with a labeling gene, Mayaji et al., Cytotechnology,4:173-180 (1990) tried to improve the expression rate and theproductivity of the recombinant, cells.

Yamaguchi et al., Biosci. Biotechnol. Biochem., 56:600-604 (1992)established serum-independent recombinant CHO sub-clones by culturingserum-dependent cells on microtiter plates as monolayer for 3 to 4 weeksin serum-free medium that contained human serum albumin, insulin andtransferrin. Approximately 0.1% of the cells were serum-independent.Part of the subclones also grew in suspension culture in serum-freemedium, yet the cells aggregated and formed lumps. The duplicating timeof the cells amounted to 1.5 days. Yet there are no data either on thestability of the serum-independent clones obtained, nor on the long timecultivation of these clones under serum-free conditions.

Media which allow for the maintenance of the metabolic activity and fora growth of cells during the serum-free phase frequently containadditional substances, e.g. growth factors, such as insulin ortransferrin, or adherence factors which substitute the serum components.

To avoid the addition of polypeptide factors, such as insulin ortransferrin, and to allow for protein free culturing conditions, varioustechniques have been developed. Thus, specifically defined, completeprotein-free media have been developed which allow for a cell-growthalso under protein-free conditions.

WO 97/05240 describes the preparation of recombinant proteins underprotein-free conditions, the cells co-expressing a growth factor inaddition to the desired protein.

JP 2696001 describes the use of a protein-free medium for the productionof factor VIII in CHO cells by adding a non-ionic surface-active agentor cyclodextrin to increase the productivity of the host cells. Toincrease the effectiveness of these additives, the addition of, e.g.,butyrate and lithium is recommended. As indicated in the specification,the addition of pluronic F-68 results in a marked increase in cellnumbers.

WO 96/26266 describes the culturing of cells in a medium which containsa glutamine-containing protein hydrolysate whose content of free aminoacids is less than 15 k of the total weight of the protein, and whosepeptides have a molecular weight of less than 44 kD. As the culturingmedium for the cell cultures, a synthetic minimum medium is used as thebasic medium to which, inter alia, fetal calf serum, gentamycin andmercaptoethanol are added in addition to protein hydrolysate. The use ofthis serum-containing medium for the recombinant production of bloodfactors has not been mentioned.

U.S. Pat. No. 5,393,668 describes special synthetic surfaces which allowfor a growth of adherent cells under protein-free conditions.

To stimulate cell proliferation, CHO cells which overexpress humaninsulin have been multiplied on an artificial substrate to which insulinis covalently bound (Ito et al., PNAS USA, 93:3598-3601 (1996)).

EP 0 872 487 describes the preparation of recombinant factor VIII inprotein-free medium containing recombinant insulin to which polyols areadded. According to the specification, the addition of pluronic F-68results in an increased factor VIII productivity of BHK cells, and theaddition of iron ions yet enhances this rise in productivity.

Reiter et al., Cytotechnology, 9:247-253 (1992) describe theimmobilization of r-CHO cells first grown in serum-containing medium ata high density on carriers, and subsequent perfusion of the immobilizedcells in protein-free medium during the production phase, wherein acontinuous liberation of protein into the cell culture supernatant wasfound. There, the cells were perfused for less than 10 generations inprotein-free medium.

Katinger et al., Adv. In Mol. Cell. Biol., 15a:193-207 (1996) describethe preparation of stable cell cultures wherein the cells areimmobilized on macroporous carriers. It is emphasized that perfusioncultures with porous carrier materials would be preferable to othermethods. Stable clones expressing recombinant proteins, such as FVIII orvon-Willebrand factor, are not described, the cells are invariably grownfirst in serum-containing medium and are only later transferred toserum- and protein-free medium.

Previous methods for the successful preparation of a large-scale cellculture under protein-free conditions have been described for continuouscell lines, in particular VERO cells (see, e.g., WO 96/15231). There,the cells are grown under serum- and protein-free conditions from theoriginal ampule up to a large technical scale of 1200 l. However, theseare not recombinant cells, but host cells which are used for theproduction of virus antigen in a lytic process.

In contrast to adherent VERO cells, e.g. CHO cells are dependent onadhesion to a limited extent only. CHO cells grown by means ofconventional methods under serum-containing conditions are capable ofbinding both to smooth and to porous micro-carriers (U.S. Pat. No.4,978,616; Reiter et al., Cytotechnology, 9:247-253(1992)). If CHO cellsare grown under serum-free conditions, they lose this property and donot adhere to smooth carriers, such as, e.g., Cytodex 3, or they detacheasily therefrom, unless adherence-promoting additives, such as, e.g.,fibronectin, are put into the medium. Because of the slight adherence ofCHO cells to carriers under serum-free conditions, the production ofrecombinant proteins thus mainly is effected in suspension culture.There, the production process may be effected as a continuous or as abatch-wise method. The recombinant cell culture at first is grown in abioreactor up to an optimum cell density, optionally the proteinexpression is induced, and for harvesting, the medium containing theexpressed proteins but also recombinant cells is withdrawn at certainintervals from the reaction tank and thus from the production process.By the continuous loss of biomass, the production efficiency in thebioreactor drops and increases again slowly only after the addition offresh medium, since the cells must grow up to the desired cell density.Thus, despite the continuous process, repeatedly there is a phase ofretardation, in which the production rate in this system drops.Furthermore, the growth and production capacity in such a system islimited by the maximum cell density attainable.

When adapting cells initially grown under serum containing conditions toprotein-free medium, it has repeatedly been found that the yield ofexpressed protein and the productivity of recombinant CHO cells greatlydrops after adaptation in protein-free medium as compared toserum-containing conditions (Paterson et al., Appl. Microbiol.Biotechnol., 40:691-658 (1994)). This is the consequence of aninstability or reduced growth of the recombinant clones due to thechanged culturing conditions. Despite the use of a stable originalclone, on account of the altered fermentation conditions, repeatedly alarge portion of the cells become cells with reduced expression or alsonon-producers, which overgrow product producers during the productionprocess, whereby the fermented culture finally largely consists ofnon-producers or of such cells having a low expression.

As a consequence, the maximum production capacity of the fermentationculture drops continuously, and a maximum product production isrestricted to a certain number of generations or cell passages.

Thus, there is a need for a system in which a continuous production ispossible over as long a period of time as possible, in particular in thelarge-scale production of recombinant proteins under serum- andprotein-free conditions.

It would furthermore be desirable to obtain a recombinant cell clonewhich is stable in the production phase for many generations underprotein free conditions and which expresses recombinant protein.

BRIEF SUMMARY OF THE INVENTION

It is the object of the present invention to provide an efficient methodof preparing recombinant proteins under serum- and protein-freecultivation and production conditions.

It is a further object to provide a stable recombinant cell clone.

It is another object of the present invention to achieve an increase inproductivity of a recombinant cell clone by using a protein- andserum-free medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the microscopy of a working cell bank of an original cloneat the time of re-adaptation from serum-containing to serum- andprotein-free medium (A), after 10 generations in serum- and protein-freemedium (B), and after 60 generations in serum- and protein free medium(C).

FIG. 2 shows the microscopy of a cell culture starting with a stablerecombinant cell clone under serum- and protein-free conditions at theworking cell bank stage (A), after 10 generations (B) and after 60generations (C).

FIG. 3 shows the results of culturing an rFVIII-CHO cell clone in a 10 lperfusion bioreactor.

a) F VIII-activity (mUnits/ml) and perfusion rate (1-5 day) over aperiod of 42 days.

b) Volumetric productivity (units factor VIII/1/day) in the perfusionbioreactor.

DETAILED DESCRIPTION OF THE INVENTION

It is the object of the present invention to provide an efficient methodof preparing recombinant proteins under serum- and protein-freecultivation and production conditions.

It is a further object to provide a stable recombinant cell clone.

It is another object of the present invention to achieve an increase inproductivity of a recombinant cell clone by using a protein- andserum-free medium.

According to the invention, this object is achieved by providing arecombinant cell clone obtainable from a cell culture that is obtainedafter culturing a recombinant original cell clone on serum-containingmedium and re-adapting the cells to serum- and protein free medium. Thecells are continued to be cultured in serum- and protein-free mediumunder production equivalent conditions for at least 40 generations.

Preferably, culturing of the cells is effected without selection for theselection labeling and/or amplification gene, e.g. in the absence of MTXin case of CHO-dhfr⁻ cells.

By original cell clone within the scope of this invention a recombinantcell clone transfectant is understood which, upon transfection of hostcells with a recombinant nucleotide sequence, expresses recombinantproduct in a stable manner under laboratory conditions. To optimizegrowth, the original clone is first grown in serum-containing medium. Toincrease the productivity, the original clone optionally is first grownin the presence of a selecting agent and selection for the selectionmarker and/or amplification marker. For the large-scale production, theoriginal cell clone is first grown under serum-containing culturingconditions up to a high cell density, and shortly before the productionphase it is re-adapted to serum- and/or protein-free medium. In thiscase, culturing is preferably effected without selection pressure.

In another preferred embodiment the recombinant original cell clone maybe cultivated in serum- and protein-free medium already from thebeginning, rendering re-adaptation unnecessary. Optionally, a selectingagent may also be used in this case, and selection may be for theselection and/or amplification marker. A respective method is, e.g.,described in EP 0 711 835.

It has been found that under these conditions, a large part of more than95% of the cells become nonproduct producers in such a cell culturewhich has been re-adapted to serum- and protein free medium. By means ofimmune fluorescence with product-specific antibodies it could be shownthat in dependence on the generation time of the cells in serum- andprotein-free medium, the number of the non-producers rises in a cultureand overgrows the product-producers, whereby the production capacity ofthe culture decreases.

The cell culture obtained after re-adaptation to serum- and protein freemedium is assayed for those cell clones of the cell population which areproducers of the stable product under serum- and protein-freeconditions, optionally in the absence of a selection pressure. This maybe effected, e.g., by means of immunofluorescence with labeledantibodies specifically directed against the recombinant polypeptide orprotein. Those cells which have been identified as product producers areisolated from the cell culture and again multiplied under serum- andprotein-free, optionally under production-equivalent, conditions.Isolation of the cells may be effected by isolating the cells andassaying for product-producers. Optimally, the cell culture containingthe stable cells is again assayed for stable recombinant clones, and thelatter are isolated from the cell culture and cloned. Subsequently, thestable recombinant cell clones obtained under serum- and protein-freeconditions are further multiplied under serum- and protein-freeconditions.

The recombinant cell clone according to the invention is characterizedin that it is stable in serum-free and protein-free medium for at least40, preferably at least 50, in particular more than 60 generations andexpresses recombinant product.

According to a particular aspect of the invention, the stablerecombinant cell clone is present in isolated form. Departing from thestable cell clone, a cell culture is obtained under serum- andprotein-free conditions by multiplying the stable cells.

The stable recombinant cell clone of the invention preferably is derivedfrom a recombinant mammalian cell. The recombinant mammalian cells maybe all cells that contain sequences which encode a recombinantpolypeptide or protein. Included are all continuously growing cellswhich grow adherently and non-adherently. Particularly preferred arerecombinant CHO cells or BHK cells. Recombinant polypeptides or proteinsmay be blood factors, growth factors or other biomedically relevantproducts.

According to the present invention, stable recombinant cell clones arepreferred which contain the encoding sequence for a recombinant bloodfactor, such as factor II, factor V, factor VII, factor VIII, factor IX,factor X, factor XI, protein S, protein C, an activated form of any oneof these factors, or VWF, and which are capable of stable expression ofthis factor over several generations. Particularly preferred arerecombinant CHO cells, which express VWF or a polypeptide having VWFactivity, factor VIII or a polypeptide having factor VIII activity, VWFand factor VIII, factor IX or factor II.

The cell clone of the invention selected under serum and protein-freeconditions is particularly characterized in that it is stable in serumand protein-free medium for at least 40, preferably for at least 50generations, particularly preferred for more than 60 generations.

To provide a master cell bank, 30 generations are required. To carry outan average batch culture on a 1,000 liter scale, at least approximately40 generations are required. Thus it has become possible for the firsttime to prepare with one individual clone a master cell bank (MCB), aworking cell bank (WCB) including approximately 8 to 10 generations andthus, a production-scale cell culture (production biomass) with up to 20to 25 generations under these conditions, since so far cell clones havebecome unstable after having grown on serum- or protein-free medium forsome generations, or have exhibited a reduced viability, whereby a) nouniform cell culture with product producers, and b) no stable productproductivity has been possible over an extended period of time.

The cell clone according to the invention thus is stable and productivefor at least 40 generations under production conditions in serum andprotein-free medium. Previously described methods merely exhibited aproduct productivity for a generation number of less than 10 generationsunder protein-free conditions (Reiter et al., (1992) supra).

The criterion for stability is held to be a minimum number of at least40 generations, preferably more than 50 generations, particularlypreferred more than 60 generations in the production process, duringwhich a stable expression of the proteins takes place and the cells donot exhibit any tumorigenic properties.

Surprisingly it has been found that the cell clone according to theinvention exhibits an increased product productivity under serum andprotein-free conditions even in comparison to the original cell clonewhich had been cultured in serum containing medium.

In addition, it has surprisingly been found that the productivity of thecultivated cells may be increased by adding additional amino acidsand/or purified, ultrafiltrated soybean peptone to the serum- andprotein-free medium. In this case, the increase in productivity is notcaused by the enhanced cell growth rate; rather, the culture conditionsdirectly influence the productivity of the cells expressing arecombinant protein.

A particular aspect is the use of a protein- and serum-free medium towhich a mixture of amino acids selected from the group of L-asparagine,L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine has beenadded.

The amino acids may be added to the medium individually or incombination.

Particularly preferred is the combined addition of all the amino acidslisted in this group, i.e. L-asparagine, L-cysteine, L-cystine,L-proline, L-tryptophan and L-glutamine.

The increase in productivity by the addition of the amino acid mixture,which may be thus achieved, was particularly surprising because thesynthetic minimum media as described in the prior art, e.g. DMEM/HAM'sF12, already contain low concentrations of amino acids.

According to a further aspect, the present invention provides a cellculture containing at least 90%, preferably more than 95%, particularlypreferred more than 98%, stable recombinant cells which are stable underserum- and protein-free conditions for at least 40 generations, inparticular for at least 50 generations, and express recombinant product.

Within the scope of the present invention, by cell culture a master cellbank (MCB), a working cell bank (WCB) or a production biomass in alarge-technical production bioreactor is understood.

According to the invention, the cell culture is particularly obtained byculturing a stable recombinant cell clone of the above-defined kindunder serum and protein-free conditions.

The cell culture of the invention is obtainable by multiplying theisolated stable cell clone from the individual clone, the seed cells upto the MCB, the WCB or a biomass on a production scale in the bioreactorunder serum and protein-free conditions, preferably without selectionpressure on the selection and/or marker gene. In particular, it has beenshown that the recombinant cells in a cell culture which are obtaineddeparting from the stable recombinant clone of the invention are stableunder serum- and protein-free conditions for at least 40 generations.

The cell culture provided according to the present invention, which hasbeen prepared from a serum and protein-independent stable cell clone,exhibits at least 90%, preferably at least 95%, particularly preferredat least 98%, stable recombinant cells under protein-free culturing andproduction conditions. By stable recombinant cells, in particularrecombinant mammalian cells are understood which are derived from thestable cell clone. Preferred are recombinant CHO cells, preferablyCHO-dhfr cells, CHO-K1 cells and BHK cells that express a blood factor,preferably recombinant vWF, factor VII, factor VIII and vWF, factor IXor factor II.

The cell culture according to the invention may contain the stablerecombinant cells in the form of a suspension culture. The cells mayalso be immobilized on a carrier, in particular on a microcarrier,porous microcarriers being particularly preferred. Porous carriers, suchas e.g. Cytoline® or Cytopore® have proved to be particularly suitable.

According to a further aspect, the present invention provides a methodfor the large-technical production of a recombinant product under serumand protein-free conditions, by using the stable cell clone according tothe invention. The method comprises the steps of providing an isolated,stable recombinant cell clone of the above-defined kind for producing acell culture. Multiplication of the isolated stable cell clone iseffected from the stable individual cell clone up to the cell cultureunder serum and protein-free conditions. In particular, alsosub-culturing of the stable cell clones is effected under protein-freeconditions, in particular without the addition of a protease, such as,e.g., trypsin. Thus it is ensured that at no time during the productionof a cell culture used in the production of a recombinant product, acontamination occurs which possibly could be caused by the addition ofserum or protein-containing additives of human or animal origin to thecell culture. Thus, for the first time a method is described whichallows for working under serum- and protein-free conditions, startingfrom the original clone, via the preparation of a working cell bank asfar as to the production biomass and the subsequent production ofrecombinant protein.

The preparation of the recombinant products with the cell culture of theinvention which contains more than 90%, preferably more than 95%,particularly preferred more than 98%, of stable product producer cells,may be effected as a suspension culture or with cells immobilized oncarriers. The process may be effected as a batch-wise or a continuousmethod or by means of perfusion technique with serum and protein freemedium.

Moreover, the culturing process may be effected by means of thechemostat method as extensively described in prior art (Wemer et al., J.Biotechnol., 22:51-68 (1992)). For example, a stirred bioreactor or anairlift reactor may be used.

The expressed recombinant proteins are then recovered from the cellculture supernatant, purified by means of methods known from the priorart, and further processed.

Any known synthetic medium can be used as the serum and protein-freemedium. Conventional synthetic minimum media may contain inorganicsalts, amino acids, vitamins and a carbohydrate source and water. Itmay, e.g., be DMEM/HAM's F-12 medium. The content of soybean or yeastextract may range between 0.1 and 100 g/l, particularly preferredbetween 1 and 5 g/l. As a particularly preferred embodiment, soybeanextract, e.g. soybean peptone, may be used. The molecular weight of thesoybean peptone can be less than 50 kD, preferably less than 10 kD.

The addition of ultrafiltrated soybean peptone having an averagemolecular weight of 350 Dalton has proven particularly advantageous forthe productivity of the recombinant cell lines. It is a soybean isolatehaving a total nitrogen content of about 9.5% and a free amino acidcontent of about 13%.

Particularly preferred is the use of a purified, ultrafiltrated soybeanpeptone having a molecular weight of ≦1000 Dalton, preferably ≦500Dalton, particularly preferably ≦350 Dalton.

Ultrafiltration may be effected by means of methods extensivelydescribed in prior art, e.g., using membrane filters with a definedcut-off.

The ultrafiltration soybean peptone may be purified by means of gelchromatography, for example using Sephadex chromatography, e.g.,Sephadex G25 or Sephadex G10 or equivalent materials; ion exchangechromatography, or size exclusion chromatography or reversed phasechromatography. These are methods well known to a skilled artisan fromprior art.

Particularly preferably a medium having the following composition isused: synthetic minimum medium (1 to 25 g/l) soybean peptone (0.5 to 50g/l), L-glutamine (0.05 to 1 g/l), NaHCO₃ (0.1 to 10 g/l), ascorbic acid(0.0005 to 0.05 g/l), ethanol amine (0.0005 to 0.05), Na selenite (1 to15 μg/l). Optionally, a non-ionic surface-active agent, such as, e.g.,polypropylene glycol (PLURONIC F-61, PLURONIC F-68, SYNPERONIC F-68,PLURONIC F-71, or PLURONIC F-108) maybe added to the medium as adefoaming agent. This agent is generally applied to protect the cellsfrom the negative effects of aeration, since without the addition of asurface-active agent, the rising and bursting air bubbles may damagethose cells which are at the surface of these air bubbles (“sparging”).(See, e.g., Murhammer and Goochee, Biotechnol. Prog., 6:142148 (1990)).

The amount of non-ionic surface-active agent may range between 0.05 and10 g/l, particularly preferred is as low an amount as possible, between0.1 and 5 g/l. Furthermore, the medium may also contain cyclodextrine ora derivative thereof. The addition of non-ionic surface-active agent orof cyclodextrine is, however, not essential to the invention.Preferably, the serum and protein-free medium contains a proteaseinhibitor, such as, e.g., serine protease inhibitors, which are suitablefor tissue culture and which are of synthetic or vegetable origin.

In another preferred embodiment the following amino acid mixture isadditionally added to the above-mentioned medium: L-asparagine (0.001 to1 g/l; preferably 0.01 to 0.05 g/l; particularly preferably 0.015 to0.03 g/l), L-cysteine (0.001 to 1 g/l; preferably 0.005 to 0.05 g/l;particularly preferably 0.01 to 0.03 g/l), L-cystine (0.001 to 1 g/l;preferably 0.01 to 0.05 g/l; particularly preferably 0.015 to 0.03 g/l).L-proline (0.001 to 1.5 g/l; preferably 0.01 to 0.07 g/l; particularlypreferably 0.02 to 0.05 g/l), L-tryptophan (0.001 to 1 g/l; preferably0.01 to 0.05 g/l; particularly preferably 0.015 to 0.03 g/l) andL-glutamine (0.05 to 10 g/l; preferably 0.1 to 1 g/l). Theabove-mentioned amino acids may be added to the medium individually orin combination. Particularly preferred is the combined addition of theamino acid mixture containing all of the above-mentioned amino acids.

In a particular embodiment a serum- and protein-free medium is usedadditionally containing a combination of the above-mentioned amino acidmixtures and purified, ultrafiltrated soybean peptone.

Surprisingly, it has proven possible to heat the medium to 70 to 95° C.,preferably 85 to 95° C., for about 5 to 20 minutes, preferably 15minutes, without causing negative effects, e.g., in order to inactivateviruses or other pathogens.

The parameters for culturing the cells, such as O₂ concentration,perfusion rate or medium exchange, pH, temperature and culturingtechnique will depend on the individual cell types used and may bedetermined by the skilled artisan in a simple manner. For instance,culturing of CHO cells may be effected in a stirring tank and underperfusion with protein-free medium at a perfusion rate of from 2 to 10volume exchanges/day, at a pH of between 7.0 and 7.8, preferably at 7.4,and an O₂ concentration of between 40% up to 60%, preferably at 50%, andat a temperature of between 34° and 38°, preferably of 37°.

Moreover, the cells may also be cultured by means of the chemostatmethod, using a pH of between 6.9 and 7.8, preferably 7.1, an O₂concentration of between 10% and 60%, preferably 20%, and a dilutionrate D of 0.25 to 1.0, preferably 0.5.

According to a further aspect, the present invention provides a methodof recovering a stable recombinant cell clone, comprising the steps of

-   -   multiplying a recombinant original clone up to the cell culture        in serum-containing medium, preferably without selection        pressure,    -   culturing the cells under serum and protein-free, preferably        under production equivalent, conditions,    -   assaying the cell culture under serum and protein free        conditions for product producers,    -   cloning the stable recombinant cell clones under serum- and        protein-free conditions, wherein cloning may be effected by        generally known techniques, such as diluting out and growing the        individual cell clones,    -   multiplying the isolated cell clones under serum and        protein-free conditions,    -   and optionally assaying the cell culture for product producers.

Only those recombinant cell clones are considered stable which expressrecombinant protein in a stable manner in protein-free medium for atleast 10, preferably at least 20, and in particular at least 50generations.

According to a further aspect, the invention relates to a method ofrecovering a stable recombinant cell clone, comprising the steps of

-   -   multiplying a non-recombinant starting cell or cell line under        serum and protein-free conditions, and cloning a stable        non-recombinant cell-clone under serum- and protein-free        conditions,    -   transfecting the stable cell clone with a recombinant nucleic        acid and isolating stable recombinant cell clones,    -   Culturing the stable cell clone transfectants in serum- and        protein-free medium, optionally under production-equivalent        conditions,    -   assaying the stable recombinant cells for production and product        stability.

A particularly preferred aspect of the present invention provides thepreparation of a stable cell clone comprising the steps of

-   -   multiplying a recombinant original clone up to the cell culture        in serum- and protein-free medium, preferably without selection        pressure;    -   culturing the cells under serum- and protein-free, preferably        under production-equivalent conditions,    -   assaying the cell culture under serum- and protein-free        conditions for product producers,    -   cloning the stable recombinant cell clones under serum- and        protein-free conditions, wherein cloning may be effected by        generally known techniques, such as diluting out and growing the        individual cell clones,    -   multiplying the isolated cell clones under serum- and        protein-free conditions, and

optionally assaying the cell culture for product producers.

Only those recombinant cell clones are considered stable which expressrecombinant protein in a stable manner in protein-free medium for atleast 10, preferably at least 20, and in particular at least 50generations.

Also preferred is the use of a serum- and protein-free medium toincrease the productivity of a cell clone expressing a recombinantprotein to which additionally a defined amino acid mixture and/orpurified, ultrafiltrated soybean peptone have been added.

The invention will be described in more detail by way of the followingexamples, as well as drawings to which, however, it shall not berestricted.

EXAMPLES Example 1 Stability of rvWF-CHO Cells After Re-Adaptation fromSerum-Containing to Serum- and Protein-Free Medium

CHO-dhfr⁻ cells were co-transfected with plasmids phAct-rvWF andpSV-dhfr, and vWF-expressing clones, as described in Fischer et al.,FEBS Letters, 351:345-348 (1994)) were sub-cloned. From those sub-cloneswhich expressed rvWF in a stable manner, a working cell bank (WCB) wasset up under serum-containing conditions, yet in the absence of MTX, andthe cells were immobilized on a porous microcarrier (Cytopore®) underserum-containing conditions. When a cell density of 2×10⁷ cells/mlcarrier matrix had been reached, readaptation of the cells to serum- andprotein-free medium was effected. The cells were continued to becultured for several generations under serum- and protein-freeconditions. By means of immunofluorescence with labeled anti-vWFantibodies, the cells were assayed in serum- and protein-free medium atdifferent points of time. The evaluation of the stability of the cellsof the working cell bank was effected prior to medium readaptation,after 10 and after 60 generations in serum- and protein-free medium.Whereas the working cell bank still had 100% rvWF producers (FIG. 1A),the portion of rvWF producers after 10 generations in serum- andprotein-free medium had decreased to approximately 50% (FIG. 1B). After60 generations, more than 95% of the cells were identified asnon-producers (FIG. 1C).

Example 2 Cloning of Stable Recombinant CHO Clones

From the cell culture containing rvWF-CHO cells according to Example 1,(the stable cell clone designated r-vWF-CHO F7 was deposited on Jan. 22,1998, at the European Collection of Cell Cultures (ECACC), Salisbury,Wilshire, SP4 0JG, UK, according to the Budapest Treaty, and receivedthe deposit number 98012206) which had been cultured for 60 generationsin serum- and protein-free medium (FIG. 1C), a dilution was made, and0.1 cells/well were each seeded in a micro-titer plate. The cells werecultured for approximately 3 weeks in DMEM/HAM's F12 without serum orprotein additions and without selection pressure, and the cells wereassayed by means of immunofluorescence with labeled anti-vWF antibody. Acell clone that had been identified as positive was used as the startingclone for the preparation of a seed cell bank. From the seed cell bank,a master cell bank (MCB) was prepared in serum- and protein-free medium,and individual ampoules were frozen off for the further preparation of aworking cell bank. Departing from an individual ampoule, a working cellbank was prepared in serum- and protein-free medium. The cells wereimmobilized on porous microcarriers and continued to be cultured forseveral generations under serum- and protein-free conditions. Atdifferent points of time the cells were assayed for their productivityin serum- and protein-free medium by means of immunofluorescence withlabeled anti-vWF antibodies. Evaluation of the stability of the cellswas effected at the working cell bank stage and after 10 and 60generations in serum- and protein-free medium. At the working cell bankstage (FIG. 2A) and also after 10 (FIG. 2B) and 60 generations (FIG.2C), approximately 100% of the cells were identified as positive stablerecombinant clones that express rvWF.

Example 3 Cell-Specific Productivity of the Recombinant Cell Clones

From defined stages during the culturing of recombinant cells, a definednumber of cells was taken and incubated with fresh medium for 24 h. ThervWF:Risto-CoF activity was determined in the cell culture supernatants.Table 1 shows that the cell specific productivity in the inventivestable recombinant cell clones was still stable even after 60generations in serum- and protein-free medium and was even higher incomparison to the original clone that had been cultured inserum-containing medium. TABLE 1 Cell specific Cell specificproductivity productivity of the after 10 working generations Cellspecific cells mU mU productivity after 60 rvWF/10⁶ rvWF/10⁶ generationsmU Cell Clone cells/day cells/day rvWF/10⁶ cells/day rvWF-CHO #808.68 5530 <10 Original cell clone r-vWF-CHO F7 62 65 60 Stable clone

Example 4 Composition of a Synthetic Serum- and Protein-Free Medium

Preferred amount (according to the knowledge at the time Component g/lof application) in g/l Synthetic minimum medium 1-100 11.00-12.00(DMEM/HAM's 12) Soybean peptone 0.5-50 2.5 L-Glutamine 0.5-1 0.36 NaHC0₃0.1-10 2.00 Ascorbic acid 0.0005-0.05 0.0035 Ethanol amine 0.0005-0.050.0015 Na-selenite 1-15 μg/l 8.6 μg/l optional: 0.01-10 0.25 SynperonicF 68

Example 5 Culturing of rFVIII-CHO Cells in Protein- and Serum-FreeMinimum Medium

A cell culture containing rFVIHi-CHO cells was cultured in a 10 lstirring tank and with perfusion. A medium according to Example 4 wasused. The cells were immobilized on a porous microcarrier (Cytopore©,Pharmacia) and cultured for at least 6 weeks. The perfusion rate was 4volume exchanges/day, the pH was at 6.9-7.2, the O₂ concentration wasapproximately 20-50%, 15 the temperature was 37° C.

FIG. 3 shows the results of culturing an rFVIII-CHO cell clone in a 10 lperfusion bioreactor.

-   -   a) F VIII-activity (mUnits/ml) and perfusion rate (1-5/day) over        a period of 42 days.

b) Volumetric productivity (units factor VIII/1/day) in the perfusionbioreactor. Cell-specific productivity Immunofluorescence (% Days ofculturing mU/10⁶ cells/day) FVIII-positive cells) 15 702 not indicated21 1125 not indicated 28 951 >95% 35 691 >95% 42 970 not indicated

Table 2 shows the stability and specific productivity of therFVIII-expressing cells. For these results, samples were taken after 15,21, 28, 35 and 42 days, centrifuged at 300 g and re-suspended in freshserum- and protein-free medium. After further 24 h, the factor VIIIconcentration in the cell culture supernatants and the cell number weredetermined. Based on these data, the specific FVIII productivity wascalculated.

A stable average productivity of 888 mUnits/10⁶ cells/day was attained.This stable productivity was also confirmed by immunofluorescence withlabeled anti-FVIII antibodies after 15, 21, 28, 35 and 42 days in serum-and protein-free medium.

Example 6 Comparison of the Productivity of Recombinant FVIII-CHO Cellsin Protein- and Serum-Free Medium Containing Additional MediumComponents

A cell culture containing rFVIII-CHO cells was cultured using a batchmethod. A medium according to example 4 was used to which the followingamino acids were added: Preferred amount (according to the knowledge atthe time of Amino acid mg/l application) in mg/l L-asparagine 1-100 20L-cysteine.HCl•H₂0 1-100 15 L-cystine 1-100 20 L-proline 1-150 35L-tryptophan 1-100 20 L-glutamine 50-1000 240

The cells were cultured at 37° C., pH 6.9-7.2. The cells were cultivatedover a period of 24-72 hours in a batch process.

The productivity of the recombinant FVIII-CHO cells was measured in thefollowing media compositions:

Mix 1 consisting of serum- and protein-free medium without soybeanpeptone and additionally containing a mixture of amino acids as listedin the above table

Mix 2 consisting of serum- and protein-free medium containing soybeanpeptone

Mix 3 consisting of serum- and protein-free medium containing soybeanpeptone and a mixture of amino acids as listed in the above table

Mix 4 consisting of serum- and protein-free medium containing, inaddition, a mixture of amino acids as listed in the above table and 2.5g/l of purified, ultrafiltrated soybean peptone. The ultrafiltratedsoybean peptone was purified by means of chromatography using aSephadex© column.

Example 7 Culturing of Recombinant FVIII-CHO Cells in Protein- andSerum-Free Medium Using Chemostat Culture

A cell culture containing rFVIII-CHO cells was cultured in a 10 lstirred bioreactor tank. A medium according to example 4 not containingsoybean peptone but containing an amino acid mixture according toexample 6 was used.

The cells were cultivated at 37° C., pH 6.9-7.2; the oxygenconcentration was in the range of 20-50% air saturation. In order todetermine the titer of factor VIII and the cell concentration in culturesupernatant, samples were taken every 24 hours. The total cellconcentration was constant from day 2 to day 14. From day 6,ultrafiltrated soybean peptone was added to the medium. The factor VIIIproductivity is measured by means of a CHROGENIX COA FVIII:c/4 system.The lack of soybean peptone in the continuous culture lead to a markeddecrease in factor VIII productivity after a few days, whereas theaddition of the soybean peptone resulted in an almost 10-fold increasein productivity. Because said addition did not increase the cell number,this clearly indicates that ultrafiltrated soybean peptone causes amarked increase in productivity, which, however, is independent of cellgrowth.

1. A method for producing a stable recombinant cell clone, which isstable under production conditions in serum- and protein-free medium forat least 40 generations, the method comprising: providing a recombinantoriginal cell clone, cultivating the recombinant original cell clone onserum-containing medium, adapting the cells to serum- and protein-freemedium in the absence of a selection pressure, testing the cell cultureafter adaptation for stable product-producers in the absence of aselection pressure and cloning a stable product-producer-cell clone inserum- and protein-free conditions in the absence of a selectionpressure.
 2. The method according to claim 1, wherein the stable cellclone obtained is present in isolated form after the step of cloning. 3.The method according to claim 1, wherein the recombinant original cellclone is a mammalian cell.
 4. The method according to claim 3, whereinthe mammal cell is selected from the group consisting of: a recombinantCHO cell and a recombinant BHK cell.
 5. The method according to claim 1,wherein the recombinant cell clone comprises a nucleic acid encoding arecombinant polypeptide or protein.
 6. The method according to claim 5,wherein the recombinant protein is a blood factor selected from thegroup consisting of: Factor II, Factor V, Factor VII, Factor VIII,Factor IX, Factor X, Factor XI, Protein S, Protein C or an activatedform of one of the factors, and vWF, and a combination thereof.
 7. Themethod according to claim 1, wherein the original recombinant cell cloneis a CHO cell expressing a recombinant polypeptide selected from thegroup consisting of: von Willebrand factor, Factor VIII, Factor IX,Factor II, and a combination thereof.
 8. A stable recombinant cellclone, obtainable by a method according to of claim 1, wherein the cellclone is stable for at least 40 generations and expresses a recombinantproduct under production conditions in serum- and protein-free medium,in the absence of a selection pressure.
 9. The cell clone according toclaim 8, wherein the clone is present in isolated form after the step ofcloning.
 10. The cell clone according to claim 8, wherein the originalrecombinant cell clone is a recombinant mammal cell.
 11. The cell cloneaccording to claim 10, wherein the mammal cell is selected from thegroup consisting of: a recombinant CHO cell and a recombinant BHK cell.12. The cell clone according to claim 8, wherein the cell comprises anucleic acid encoding a recombinant polypeptide or protein.
 13. The cellclone according to claim 12, wherein the recombinant protein is a bloodfactor selected from the group consisting of: Factor II, Factor V,Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Protein S,Protein C or an activated form of one of the factors, vWF, and acombination thereof.
 14. The cell clone according to claim 12, whereinthe recombinant protein is Factor VIII.
 15. The cell clone according toclaim 12, wherein the cell clone is a recombinant CHO cell.
 16. The cellclone according to claim 15, wherein the cell clone expresses arecombinant polypeptide selected from the group consisting of: vonWillebrand factor, Factor VIII, Factor IX, Factor II, and a combinationthereof.
 17. The cell clone according to claim 15, wherein the cellclone co-expresses Factor VIII and vWF.
 18. A cell culture obtainable bycultivating a stable recombinant cell clone according to claim
 12. 19.The cell culture of claim 18, wherein the cell culture contains at least90% stable recombinant cell clones.
 20. The cell culture according toclaim 19, wherein the stable recombinant cell clones are mammaliancells.
 21. The cell culture according to claim 20, wherein the mammaliancells are recombinant CHO cells.
 22. The cell culture according to claim21, wherein the CHO cells are selected from the group consisting of:CHO-DHFR cells and CHO-K1 cells.
 23. The cell culture according to claim18, wherein the stable recombinant cells comprise a nucleic acidencoding a recombinant polypeptide or protein.
 24. The cell cultureaccording to claim 18, wherein the stable recombinant cells areimmobilised on a microsupport.
 25. A method for industrial production ofa recombinant product under serum- and protein-free conditions, themethod comprising: providing an isolated, stable recombinant cell cloneaccording to claim 8, propagating the stable cell clone in serum- andprotein-free medium from the initial clone up to the cell culture in theabsence of a selection pressure, producing the cell culture containingstable cells in the bioreactor in the absence of a selection pressure,and harvesting the recombinant product from the culture supernatant. 26.The method according to claim 25, wherein the serum- and protein-freemedium is a synthetic minimal medium comprising an extract selected fromthe group consisting of: a yeast extract and a soy extract.
 27. Themethod according to claim 25, wherein the medium contains cyclodextrinor a derivative thereof.
 28. The method according to claim 25, whereinthe serum- and protein-free medium contains a protease inhibitor. 29.The method according to claim 25, wherein the recombinant product is aprotein.